Growth Factors. Growth factors can be broadly defined as multifunctional, locally acting intracellular signaling polypeptides which control both the ontogeny and maintenance of tissue form and function. The protein products of many proto-oncogenes have been identified as growth factors and growth factor receptors.
Growth factors generally stimulate target cells to proliferate, differentiate and organize in developing tissues. The action of growth factors is dependent on their binding to specific receptors that stimulate a signaling event within the cell. Examples of growth factors include platelet derived growth factor (PDGF), insulin like growth factor (IGF), transforming growth factor beta (TGF-β), transforming growth factor alpha (TGF-α), epidermal growth factor (EGF) and connective tissue growth factor (CTGF). Each of these growth factors has been reported to stimulate cells to proliferate.
Connective Tissue Growth Factor. CTGF is a cysteine rich monomeric peptide a molecular weight of about 38 kd. As previously reported, CTGF has both mitogenic and chemotactic activities for connective tissue cells. CTGF is secreted by cells and is believed to be active upon interaction with a specific cell receptor.
CTGF is a member of a family of growth regulators which include, for example, mouse (fisp-12) and human CTGF, Cyr61 (mouse), Cef10 (chicken), and Nov (chicken). Based on sequence comparisons, is has been suggested that the members of this family have a modular structure consisting typically of at least one of the following: (1) an insulin-like growth factor domain responsible for binding; (2) a von Willebrand factor domain responsible for complex formation; (3) a thrombospondin type I repeat, possibly responsible for binding matrix molecules; and (4) a C-terminal module found in matrix proteins, postulated to be responsible for receptor binding.
The sequence of the cDNA for human CGTF contains an open reading frame of 1047 nucleotides with an initiation site at about nucleotide 130 and a TGA termination site at about nucleotide 1177, and encodes a peptide of 349 amino acids. The cDNA sequence for human CTGF has been previously disclosed in U.S. Pat. No. 5,408,040.
The CTGF open reading frame encodes a polypeptide which contains 39 cysteine residues, indicating a protein with multiple intramolecular disulfide bonds. The amino terminus of the peptide contains a hydrophobic signal sequence indicative of a secreted protein and there are two N-linked glycosylation sites at asparagine residues 28 and 225 in the amino acid sequence.
The synthesis and secretion of CTGF are believed to be selectively induced by TGF-β and BMP-2, as well as potentially by other members of the TGF-β superfamily of proteins. As reported in the art, although TGF-β can stimulate the growth of normal fibroblasts in soft agar, CTGF alone cannot induce this property in fibroblasts. However, it has been shown that the synthesis and action of CTGF are essential for the TGF-β to stimulate anchorage independent fibroblast growth. See, e.g., Kothapalli et al., 1997, Cell Growth & Differentation 8(1):61-68 and Boes et al., 1999, Endocrinology 140(4):1575-1580.
With respect to biological activity, CTGF has been reported to be primarily mitogenic in nature (able to stimulate target cells to proliferate). CTGF has also been reported to have chemotactic activity. Pathologically, the full-length CTGF molecule has been reported to be involved in conditions where there is an overgrowth of connective tissue cells and overdeposition of the extracellular matrix. CTGF has also been described in the art to be associated with conditions relating to vascular endothelial cell migration and proliferation, and neovascularization. The diseases and disorders relating to these conditions, include, for example, fibrosis of the skin and major organs, cancer, and related diseases and disorders such as systemic sclerosis, angiogenesis, atherosclerosis, diabetic nephropathy, and renal hypertension. (See, e.g., Toshifumi et al, 1999, Journal of Cellular Physiology 18191):153-159; Shimo et al., 1999, Journal of Biochemistry 126(1):137-145; Murphy et al., 1999, Journal of Biological Chemistry 274(9):5830-5834; Wenger et al., 1999, Oncogene 18(4):1073-1080; Frzier et al., 1997, International Journal of Biochemistry & Cell Biology 29(1);153-161; Oemar et al., 1997, Circulation 95(4);831-839.)
CTGF has also been reported to be useful in wound healing and repair of connective tissue, bone and cartilage. In this aspect, CTGF has been described as an inducer of bone, tissue, or cartilage formation in disorders such as osteoporosis, osteoarthritis or osteochondrytis, arthritis, skeletal disorders, hypertrophic scars, bums, vascular hypertrophy or wound healing. (See, e.g., U.S. Pat. No. 5,837,258; Ohnishi et al., 1998, Journal of Molecular and Cellular Cardiology 30(11):2411-2422; Nakanishi et al., 1997, Biochemical and Biophysical Research Communications 234(1):206-210; Pawar et al., 1995, Journal of Cellular Physiology 165(3):556-565.)
In summary, CTGF has been implicated in numerous fibrotic and cancerous conditions, and has been described to contribute to wound healing. As a result, there is a need in the art to identify useful methods of modulating the activity of CTGF to treat these various diseases and disorders. Prior to the present invention, there has been no report that regions or domains of CTGF are responsible for signaling different biological activities. Moreover, prior to the instant invention, there has been no disclosure of treating diseases and disorders associated with cell proliferation and/or the overproduction of the extracellular matrix by inhibiting the biological activity of a specific region or domain of CTGF.